This proposal is for a five year extension of a Merit Award to build on the considerable progress made during the current funding period. Focusing on the most exciting aspect of our previous work, we will concentrate on the following three areas. AIM 1. Expression Analysis of Colorectal Cancer. One of our most interesting discoveries showed substantial and unexpected differences in allelic expression in normal and disease states. Specifically, we demonstrated that decreased expression from one allele of APC was sufficient to cause disease and that variation in allelic expression was common in the general population. However, the extent to which inherited and somatically acquired variations in allelic expression contribute to human neoplasia is largely unknown. To address this issue, we will perform a comprehensive evaluation of allelic expression in human colorectal cancer. This study will help define the extent and basis of altered allelic expression in human.cancer. Those transcripts displaying altered allelic expression due to silencing by methylation, nonsense mediated decay or promoter mutations should prove particularly interesting and will be the subject of further investigation. AIM 2. Copy Number Analysis of Colorectal Cancer. Some of our most important discoveries were made using new approaches for the genetic analyses of cancer. In this aim, we will combine Digital Karyotyping, SAGE and high throughput mutational analyses to define the targets of amplification events in colorectal cancers. We will focus our initial analysis on amplified genes in order to identify oncogenes which generally have better properties for therapeutic and diagnostic applications. AIM 3. Molecular Characterization of Tumor Anqiogenesis. Our discoveries of the TEMs have been particularly revealing with regards to tumor angiogenesis. The TEM1 KO mice provided new insights into the role of stromal tissue in tumor growth and invasion. Likewise, our identification of ligands for TEM7 and TEMS revealed unexpected associations. We will continue our work on this aim by generating and characterizing TEMS and TEMS KO mice. We will also use a yeast two hybrid and an affinity purification screen to identify TEMS ligands. The above studies will provide additional insights into the role of the TEMs in tumor angiogenesis and growth as well as potentially provide leads to the therapeutic applications of the TEMs. Overall, the proposed studies should provide important new insights into tumor angiogenesis (Aim 3) and the specific targets (Aims 1 & 2) of genetic alterations in human cancer. Relevance to Public Health: The most exciting recent advances in the management of cancer have been based on a molecular and genetic understanding of this deadly disease. The studies proposed in this application are directed at defining the molecular basis for the recruitment of blood vessels by tumors and at identifying the genetic alterations that drive cancer development. Both these avenues should help drive new therapeutic approaches and the genetic alterations should have immediate applications to early diagnosis.